This invention describes a novel way to harvest and produce AAV.
Adeno-associated virus (AAV), a member of the Parvovirus family, is a small nonenveloped, icosahedral virus with a single-stranded linear DNA genome of 4.7 kilobases (kb) to 6 kb. AAV is assigned to the genus, Dependovirus, because the virus was discovered as a contaminant in purified adenovirus stocks. AAV's life cycle includes a latent phase at which AAV genomes, after infection, are integrated into host genomes and an infectious phase in which, following either adenovirus or herpes simplex virus infection, the integrated AAV genomes are subsequently rescued, replicated, and packaged into infectious viruses. The properties of non-pathogenicity, broad host range of infectivity, including non-dividing cells, and integration make AAV an attractive delivery vehicle.
A variety of different AAV sequences and methods for isolating same from tissues have been described. AAV1-6, AAV7, AAV9 and AAV9, amongst other AAV sequences obtained from simian or human tissue sources have been described. See, e.g., International Patent Publication Nos. WO 02/33269, WO 02/386122 (AAV8), and International Patent Publication No. WO 2005/033321. With this, a move away from defining AAV strictly by serologic cross-reactivity (serotypes) has occurred. Recent literature defines the relationship between these AAV in terms of phylogenetic relatedness, proposing groups termed “clades”. See, e.g., Gao et al, J Virol, 78(12):6381-6388 (June 2004); International Patent Publication No. WO 2005/033321.
Current methodology for production of AAV has been founded largely in view of the observation that AAV2 is cell-associated and thus, thought to reside primarily in the producing cells. Therefore most current state-of-the-art AAV production strategies obtain vector particles from the cellular pellet of the production cell line. Each of these strategies employs some methodology of releasing vector from the cell pellet by sonication, enzymatic, chemical or physical lysis. This unfortunately releases all intracellular proteins and debris into the viral harvest. Therefore the subsequent purification procedure is more demanding. Because of the relatively low efficiency of both production and purification, it is necessary to start with a large amount of producing cells.
What are needed are efficient methods of production and purification of AAV.